Both U.S. licensed influenza vaccines provide little heterosubtypic protection, and thus, must be reformulated annually in an attempt to match the vaccine strains with those circulating in subsequent epidemics. An ideal influenza vaccine adjuvant would induce significant heterosubtypic protection. Preliminary studies suggest that the cationic lipid DNA complex (CLDC), consisting of non-coding DNA encapsulated in cationic lipids, complexed with inactivated influenza viruses induces robust antibody and T- cell immunity, and confers cross-protection from mismatched influenza strains. I propose to elucidate the mechanisms of CLDC as an adjuvant by determining the identity of the immune cells activated by CLDC and intracellular signaling pathways required for CLDC's effects. First, I will identify if CLDC is internalized by dendritic cells (conventional, cDCs and plasmacytoid, pDCs) or macrophages in vivo. My preliminary studies suggest that these cell types uptake CLDC in vitro. I will then determine the impact of depletion of specific immune cells on CLDC's adjuvant effects. My second aim will define the role of Toll-like receptor (TLR) signaling in CLDC adjuvant immune responses. My preliminary studies show that CLDC is retained in early endosomes, suggesting that activation of the endosomal-located TLR9 (stimulated by unmethylated CpG motifs) may mediate CLDC's immunostimulatory effects. I will investigate the role of TLRs in CLDC's adjuvant responses using mice genetically deficient in TLR signaling. Lastly, I will examine the importance of type I interferons (IFN) in CLDC's adjuvant responses. Preliminary studies suggest that CLDC stimulates both cDCs and pDCs to produce robust levels of IFN-? in vitro. I will use mice lacking the type I IFN receptor to determine the importance of these cytokines in CLDC's adjuvant responses. If either TLR signaling or type I IFNs are important in CLDC's responses, subsequent experiments will examine which cell types require these signals. Overall, these studies will provide important mechanistic insights into how CLDC can confer heterosubtypic protection when used as an adjuvant for an influenza vaccine. PUBLIC HEALTH RELEVANCE: Influenza infections cause substantial morbidity and mortality worldwide, thus a major goal of influenza vaccine research is to develop vaccines that provide robust, cross-protective immune responses. Preliminary studies suggest that the cationic lipid DMAcomplex (CLDC) plus inactivated influenza viruses induces strong cellular immune responses in mice, which has generate interest for the use of CLDC as a novel and successful vaccine adjuvant. My proposed studies will provide important mechanistic insights into CLDC's immunostimulatory effects and may contribute to the design of a more effective influenza vaccine.